MARINE ECOLOGY PROGRESS SERIES Vol. 183: 169-178.1999 Published July 6 1 Mar Ecol Prog Ser l Distributions of total and active bacteria in biofilms lining tubes of the onuphid polychaete Diopatra cuprea Tina M. Phillips, Charles R. Lovell* Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208, USA ABSTRACT: Infaunal burrows and tubes support elevated levels of microbial biomass and activities relative to the surrounding bulk sediments. The impacts of these structures on sediment biogeochem- istry have been intensively studled, but very little is known about the microbiota or their spatial orga- nization within these microenvironments. Distributions of bacterial cells and microcolonies and of potentially active bacterial cells in tubes of the onuphid polychaete Diopatra cuprea were determined using confocal scanning laser microscopy. Distributions were examined among different tubes, at dif- ferent locations within a tube, and at different depths in the biofilms lining these tubes. The average quantity of cells across all tubes examined was 5.61 X 10' cm-3 and there was no trend in the distribu- tion of cells along the length of the tube or with increasing depth in the biofilm. Cell size across all tubes collected in February 1997 averaged 0.085 pm3 but varied seasonally. Mean cell size increased with increasing depth below the sediment surface of the tube site sampled, and decreased with increasing depth in the biofilm. Microcolonies were abundant and not confined to specific depths within the biofh or locations within the tube. Potentially active cells were observed at all depths within the biofilm and at least 46% (mean = 68%) of cells at any given site were potentially active. We hypothe- size that the observed trends in cell distribution are responsive to tube irngation by the host polychaete KEY WORDS: Microbial communities . Infaunal burrows . Spatial distributions . Confocal scanmng laser microscopy INTRODUCTION 1980, Grant 1983).Sediment mixing can greatly stimu- late some microbial activities by increasing the quanti- Geochemical cycling of the major biogenic elements ties of potential electron donors and acceptors below in nearshore marine sediments is primarily mediated the sediment-water interface (Findlay & White 1983, by microorganisms. However, sediments are far from 1984, Krantzberg 1985, Findlay et al. 1990). Mixing homogeneous and some specific microenvironments also disrupts chemocline microbial communities and are particularly conducive to microbial activities. may significantly alter the interactions among various Chemocline environments, such as those found at the microbial functional groups and affect their potential sediment-water interface, are well known to be sites of for consortial metabolism (Paerl & Pinckney 1996). enhanced microbial activities (Novitsky 1983, Novitsky Macrofaunal burrows and tubes also provide signifi- & Karl 1986).The classic model of these interface envi- cant chemocline niches for microbial growth. These ronments places aerobic metabolism at the sediment structures are dug into anoxic subsurface sediments, surface, followed by various anaerobic processes in irrigated with oxic seawater, and provide a large sur- deeper sediment layers (e.g. Fenchel 1969, Revsbech & face area for diffusive exchange (Aller & Yingst 1978, Jorgensen 1986, Aller 1988). However, surface sedi- Aller 1980, Boudreau & Marinelli 1994). Irrigation of ments are subject to high levels of disturbance due to the burrow by the macrofaunal host organism results physical and biogenic reworking (Yingst & Rhoads in the formation of radial chemoclines extending out from the burrow wall into the surrounding sediments 'Addressee for correspondence. E-mall: [email protected] (Aller & Yingst 1978, Aller 1983, 1988, Boudreau & O Inter-Research 1999 Resale of full article not permitted 170 Mar Ecol Prog Ser 183: 169-178, 1999 burrow lining biofilms differs from that of adjacent bulk sediments (Dobbs & Guckert 1988, Steward et al. 1996) and that burrow and tube microbiota play impor- tant roles in the mineralization of organic compounds and the geochemical cycling of biogenic elements (e.g. Kristensen et al. 1985, 1991, Binnerup et al. 1992).The rates of geochemical processes within the burrow biofilm rely mainly on the constituents of the microbial community and their levels of activity. The latter are obviously hlghly dependent on the properties of the burrow (stability and permeability) and on the irriga- tion and exudation activities of the host animal. How- ever, the distributions and potential for consortial interactions of these organisms are almost completely unknown and can best be studied using microscopic analysis. Recent developments in microscopy, specifically confocal scanning laser microscopy, allow examination of bacterial biofilms in a non-invasive manner (Coster- ton et al. 1995). Studies to date have primarily exam- ined biofilms grown on impermeable surfaces and little is known about natural biofilms growing on permeable surfaces and the rnicrobiota inhabiting them. In this study, we examined distributions of total and active bacterial cells in biofilms lining the stable tube struc- ture constructed by a ubiquitous infaunal polychaete, Diopatra cuprea. Several key features of the tube lin- ing biofilm microbiota were revealed. Fig. 1. Diopatra cuprea tube structure and the sampling pro- cedure used to determine distributions of bacteria in D. cuprea tube lining biofilms. For each tube, two 8 cm segments CHARACTERISTICS OF THE DIOPATRA CUPREA of the unreinforced portion (Segments A and B) were col- lected. In each of the segments, 5 sites (1to 5) were examined. TUBE At each site, confocal images were collected at 1 pm depth intervals from the surface of the biofilm to the tube wall The Diopatra cuprea tube is constructed from a mucous, sulphated polysaccharide, released from secreting glands, which hardens when it comes in con- Marinelli 1994, Marine111 & Boudreau 1996). These tact with seawater (Myers 1972).Each tube displays 2 radial chemoclines provide overlapping gradients of types of construction, reinforced and unreinforced potential electron donors and acceptors and are stable (Fig. 1).The reinforced section rises from the sediment over time spans long enough for development of surface and can also extend down into shallow subsur- complex microbial communi.ties (Aller & Aller 1986, face sediments. The length of this section is dependent Marinelli et al. unpubl.). Macrofaunal burrows vary on the degree of sediment disturbance, and the poly- in their structural properties, ranging from loosely chaete will extend it if the exposed portion becomes packed sediment/mucopolysaccharide aggregates, as buried. The reinforced section of the tube is decorated seen in the burrows of the terebellid polychaete with debris (small pebbles, shell fragments, and plant Amphjtnte ornata, to well-defined polysaccharide detritus) from the sediment surface. Occasionally, tubes and/or proteinaceous tubes, such as those of the will have 2 reinforced openings. The unreinforced sec- chaetopterid polychaete Chaetopterus variopedatus tion occurs below the sediment surface and is con- and the onuphid polychaete Diopatra cuprea (Myers structed as the worm moves vertically down through 1972, Aller & Yingst 1978, Aller 1983).These structures the sediments, creating a mucous sheath. The only for- promote development of microbial biofilms containing eign materials that may be attached to this portion of high levels of microbial biomass (Steward et al. 1996) the tube are sediment particles in direct contact with and supporting intense geochemical cycling activities the mucus when it is secreted. Additional layers of (Kristensen et al. 1985, 1991, Binnerup et al. 1992). mucus are laid within the tube over time, so the thick- Previous studies have shown that the microbiota of ness of the tube is hrectly proportional to its age. Phillips & Lovell: Bacterial distributions in Djopatra cuprea tubes 171 MATERIALS AND METHODS These samples were transported to Columbia, SC, and held in a recirculating sea water tank overnight. Sampling site. Diopatra cuprea tubes were collected Sample preparation and staining procedures. Each from an intertidal sandflat located at Oyster Landing, of the 10 tube segments from February 1997 was in the North Inlet saltmarsh (33" N, 79" W), near secured onto a glass microscope slide with rubber Georgetown, South Carolina, USA. Oyster Landing is a bands approximately 5 cm apart. The 2 cm ends desig- small tidal tributary bordered by Spartina alterniflora nated for handling were then removed. The portion of marsh and oyster beds (Allen et al. 1992, Lovell & the 8 cm segment between the 2 rubber bands was cut Piceno 1994). The sandflat from which samples were longitudinally and unrolled with the inner tube surface collected is completely exposed during low tide and facing up. These samples were stained for enumera- consists of quartz sand (median grain size of 376 pm) tion and measurement of total cells and microcolonies. containing 0.22% silt and clay, and 0.83% organic SYTOX Green was selected as the primary fluores- matter, by dry weight (Lovell & Piceno 1994). cent stain after substantial background fluorescence Sample collection. All Diopatra cuprea tubes col- problems were encountered with acridine orange and lected for this study were built by solitary individuals fluorescein isothiocyanate. DAPI (4,6-diamidino-2- and had no tube branches. The amount of debris